BD Phoenix Research Articles

EXTENDED SPECTRUM OF B- LACTAMASE STATUS IN ESCHERICHIA COLI ISOLATED FROM URINARY TRACT INFECTIONS IN DUHOK CITY- IRAQ

Objectives: To study the pattern of ESβL E. coli in cross sectional study from urine of patients in Azadi and Heve Paediatric Teaching Hospitals in Duhok city. Methods: This study was conducted between June 2016 and December 2016. A study of 325 of E.coli isolates from urine samples in Azadi Teaching Hospital and 420 isolates from Heve Teaching Hospital. Confirmation of ESβL production was performed using double-disk synergy test and the BD Phoenix automated system.. Results: There were 175 (53.8%) confirmed strains of ESβLs E. coli among adults from Azadi Hospital and 163 (38.8%) strains among paediatrics from Hevi Hospital which were tested by double-disk synergy test and BD Phoenix. There was no statistical significance for ESβL strains regarding the gender of the patients. Majority of ESβL E. coli were among 7-9 years and 25-65 years. The rate of antibiotic resistance was > 90% for Ceftazidime, Ceftriaxone, Cefixime, Cefepime, Augmentin and Aztreonam for both adults and paediatric age groups. The rate of sensitivity for Imipenem, Amikacin and Nitrofurantoin was 4.5%, 8% and 16% respectively for adults and 17% 15.5% and 24.5% for paediatrics respectively.

1192. Identification of a Novel Enterobacter cloacae Isolate Producing an IMP-13 Metallo-β-Lactamase

BackgroundMetallo-β-lactamases (MBLs) have been identified as emerging resistance determinants in Enterobacteriaceae, A. baumanii, and P. aeruginosa. Early identification of carbapenemase-producing organisms (CPOs) is essential to prevent dissemination within healthcare settings. We report a case of a patient who was blood culture positive for a multidrug-resistant E. cloacae which was subsequently found to be positive for the MBL blaIMP-13.MethodsA 74-year-old female, with no significant past medical or travel history, developed sepsis 2 days after undergoing debulking surgery for stage IIIc ovarian carcinoma. Blood cultures were positive for Gram-negative bacilli and the organisms identified as Enterobacter spp. with blaIMP MBL (Verigene). Antimicrobial susceptibility testing demonstrated high-level resistance to all penicillins, ureidopenicillins, cephalosporins, and β-lactam/inhibitor antibiotics, and susceptibility to colistin, tigecycline, and monobactams.ResultsFurther testing using micro-broth dilution, BD phoenix, and Etest, demonstrated susceptible MICs to meropenem and imipenem, with intermediate to resistant MICs to ertapenem. The patient was treated with a combination therapy of amikacin, aztreonam, and ceftazidime-avibactam and responded clinically. Per standard protocol, the organism was sent to WI Laboratory of Hygiene for further characterization. Phenotypic testing using the modified carbapenem inactivation test (mCIM) was positive, indicating the presence of a carbapenemase; however, results using Xpert CarbaR (Cepheid) were negative. Subsequent sequencing of the isolate confirmed the presence of blaIMP-13.ConclusionThis was an important case for several reasons. First, blaIMP-13 is historically reported in Pseudomonas aeruginosa. Indeed, this was the first report of Enterobacteriaceae harboring blaIMP in WI. Second, it had unique susceptibility pattern to carbapenems and was not detected by the CarbaR. Third, these data demonstrate clinical success in treating an MBL CPO with a combination anti-microbial regimen, based on an understanding of resistance mechanisms involved. This report calls for more vigilant screening for CPO using both phenotypic and genotypic methods.Disclosures N. Ledeboer, Luminex: Consultant, Consulting fee.

1172. Travel-Associated Multidrug-Resistant Organism Acquisition and Risk Factors Among US Military Personnel

BackgroundInternational travel is a risk factor for incident colonization with extended spectrum β-lactamase (ESBL)-producing organisms. These and other multidrug-resistant (MDR) bacteria are major pathogens in combat casualties. We evaluated risk factors for colonization with MDR bacteria in US military personnel traveling internationally for official duty.MethodsTravMil is a prospective observational study enrolling subjects presenting to military travel clinics. We analyzed surveys, antimicrobial use data, and pre- and post-travel self-collected perirectal swabs in military travelers to regions outside the continental United States, Canada, Western or Northern Europe, or New Zealand presenting to one clinic from December 2015 to December 2017. Gram-negative isolates recovered from swabs underwent real-time identification and susceptibility testing (BD Phoenix). Characteristics of trip and traveler were analyzed to determine risk factors for MDR organism colonization.ResultsOne hundred ten trips were planned by 99 travelers (74% male, median age 38 years [IQR 31, 47.25]); 72 trips were completed by 64 travelers. Median trip duration was 21 days (IQR 12.75, 79.5). Of those with trips completed, 17% traveled to Mexico/Caribbean/Central America, 15% to Asia, 57% to Africa, and 10% to South America; 56% stayed in hotels and 50% in dormitories/barracks. Travelers used doxycycline (15%) for malaria prophylaxis, 11% took an antibiotic for travelers’ diarrhea (TD) treatment (fluoroquinolone 7%, azithromycin 4%). Incident MDR organism colonization occurred in eight travelers (incidence density 3.5/1,000 travel days; cumulative incidence 11% of trips [95% CI: 4%–19%]), all ESBL-producing E. coli. A higher incidence of ESBL-producing E. coli acquisition was associated with travel to Asia (36% vs. 7%, P = 0.02) but not with travel to other regions, TD, or use of antimicrobials. No relationship was seen between fluoroquinolone or doxycycline exposure and resistance to those antimicrobials.ConclusionConsistent with other studies of US military personnel travelers, incident colonization with MDR organisms following official travel occurs at a lower rate in this population compared with civilian travelers, with no identified modifiable risk factors. The highest incidence of ESBL acquisition was observed during travel to Asia.Disclosures All authors: No reported disclosures.

1184. Resistance Patterns and Susceptibility Analysis of Klebsiella pneumoniae Infections in Service Members Who Sustained Trauma in Iraq and Afghanistan

Background Klebsiella pneumoniae was the third most common species of multidrug-resistant (MDR) Gram-negative organism in military trauma patients injured in Iraq and Afghanistan (2009–2014). This study aims to characterize the antimicrobial susceptibility and resistance patterns of K. pneumoniae isolates in these patients.MethodsAll infecting K. pneumoniae isolates (IKpI) archived by the Trauma Infectious Disease Outcomes Study (TIDOS) and 96 colonizing isolates (CKpI) from groin swabs were included (6/09-12/14). All CKpI linked with IKpI were included; the remainder to total 50 MDR and 46 non-MDR CKpI were chosen randomly. Antimicrobial identification and susceptibilities were determined by CLSI criteria using the BD Phoenix Automated Microbiology System. MDR was defined as either resistance to ≥3 classes of aminoglycosides, β-lactams, carbapenems, and/or fluoroquinolones or production of an ESBL or KPC.ResultsOf 588 K. pneumoniae archived isolates, 237 isolates were included in the analysis (141 IKpI and 96 CKpI). IKpI sources were 40% wound, 22% respiratory, 20% blood, 9% urine, and 9% other. Antibiotic susceptibilities for IKpI were: cefazolin (CFZ) 20%, ceftriaxone 30%, levofloxacin 62%, piperacillin–tazobactam (PTZ) 41%, meropenem 96%, and amikacin 89%. MDR IKpI and CKpI were more likely to have had prior fluoroquinolone (82% vs. 18%, P < 0.01) or anti-pseudomonal penicillin (53% vs. 47%, P < 0.01) exposure. Seventeen patients had CKpI cultured at a median of 5 days (IQR 2–17) before a subsequent IKpI with 11 (65%) having MDR CKpI. All IKpI isolated after MDR CKpI were also MDR. Among IKpI recovered after non-MDR CKpI, new resistance was noted in 1 IKpI to gentamicin (200 days post-CKpI), 1 IKpI to ertapenem (7 days post-CKpI), two IKpI to CFZ (10 days and 17 days, respectively), and 1 IKpI to PTZ (19 days post-CKpI). Serial isolates of IKpI had similar MDR status (63% initial IKpI were MDR, whereas 76% of subsequent IKpI were MDR).Conclusion K. pneumoniae isolates in military trauma patients from Iraq and Afghanistan had challenging resistance patterns. Prior exposure to fluoroquinolones and anti-pseudomonal penicillins were associated with MDR K. pneumoniae isolation. MDR status of CKpI predicted subsequent IKpI MDR status.Disclosures All authors: No reported disclosures.

1198. Clinical Characteristics and Outcomes of Klebsiella pneumoniae Infections in Service Members Who Sustained Trauma in Iraq and Afghanistan

Background Klebsiella pneumoniae infections present a challenge to the clinician due to increasing resistance. K. pneumoniae was the third most common species of multidrug-resistant (MDR) Gram-negative organism in trauma patients sustaining injuries in Iraq and Afghanistan from 2009 to 2014. This study aims to elucidate the epidemiology of these infections by characterizing clinical aspects, risk for MDR infections, and outcomes.MethodsAll initial and serial (≥7 days from prior isolate) infecting K. pneumoniae isolates were collected from the Trauma Infectious Disease Outcomes Study (TIDOS) (6/09-12/14). Antimicrobial susceptibilities were determined using the BD Phoenix Automated Microbiology System and CLSI criteria. MDR was defined as either resistance to ≥3 classes of aminoglycosides, β-lactams, carbapenems and/or fluoroquinolones or production of an ESBL or KPC.ResultsOf 588 K. pneumoniae isolates in the TIDOS registry, 141 infecting isolates (98 initial) from 51 patients met inclusion criteria. Initial isolates were respiratory (31%), wound (25%), blood (20%), urine (10%), intra-abdominal (8%) and other (6%). All patients were male with a median age of 23 years (IQR 21–28). The majority of patients (82%) suffered blast injuries; of which, 88% were from improvised explosive devices. Patients had a median injury severity score (ISS) of 38 (IQR 30–45) and time from injury to first infecting K. pneumoniae isolate was 15 days (IQR 8–31). The median hospital stay was 49 days (IQR 28–70) and four patients died. All patients had received antibiotics prior to diagnosis. Twenty-three (46%) patients had initial isolates classified as MDR. There was no difference in age, ISS, or time from injury to first isolation among those who did and did not have initial MDR isolates. Sixteen patients had 64 serial isolates, of which 24 were wound, 20 respiratory, 14 blood and six urine. Three of these 16 patients died compared with 1 of 35 patients without serial isolates.Conclusion K. pneumoniae infections are common among combat casualties. Patients with K. pneumoniae infections were severely injured and almost half of initial infecting isolates were MDR, complicating treatment.Disclosures All authors: No reported disclosures.

2071. Evaluation of BD Phoenix™ CPO Detect Assay for Detection of Carbapenemase Producing Organisms in Clinical Samples in Singapore

BackgroundRapid and accurate detection of CPO is crucial to a targeted infection control strategy, as in Tan Tock Seng Hospital (TTSH), a large tertiary hospital in Singapore, where cohorting of CPO colonised patients is driven by PCR-based genotypic identification. A newly released panel for the BD Phoenix system, the CPO Detect panel, includes CPO detection with Amber Class identification, alongside standard Phoenix antibiotic susceptibility testing. We evaluated this system in the context of the TTSH CPO control strategy.MethodsA total of 201 isolates from CHROMID® positive rectal swabs taken as part of inpatient screening, and from clinical samples with confirmed carbapenem resistance, were assayed prospectively between January and April 2018. Ninety-five samples were sampled retrospectively from 2017. CPO genotype was determined using PCR targeting NDM, KPC, oxa48-like, IMI and IMP carbapenemases. Isolates were analysed on the CPO Detect assay in parallel.ResultsA broad range of CPO genotypes was achieved and results were comparable in both prospective and retrospective samples. Overall, a concordance of 76% was found between CPO Detect determination of CPO status (both positive and negative) and PCR (238/313 isolates). PCR genotype was in agreement with the Ambler class found by CPO Detect in 151/200 positives (75.5%), 27 samples were not assigned an Ambler class and Ambler class was mismatched in 8 samples. Partial agreement was noted in 17 samples in which CPO Detect indicated a single Ambler class, but PCR identified two carbapanemase genes. CPO Detect outright failed to detect 14/200 PCR positive samples (7%) of which 10 were IMI. CPO Detect did however identify a CPO in a further 54 samples which were PCR negative.ConclusionCompared with PCR, CPO Detect had a sensitivity of 93% in CPO detection and agreement of 75.5% with respect to Ambler class specificity. False negatives were overwhelmingly the IMI genotype. We are continuing to characterise these by further molecular means, as well as the 54 samples found by CPO Detect but PCR negative. Disclosures All authors: No reported disclosures.

COMPARATIVE STUDY OF GELATINASE ACTIVITY AND PELLICLE FORMATION AMONG EXTENDED-SPECTRUM BETA-LACTAMASE AND NON-EXTENDED-SPECTRUM BETA-LACTAMASE PRODUCING ACINETOBACTER BAUMANNII FROM DIABETIC FOOT ULCER INFECTIONS

Objective: The aim of the study was to assess and compare the gelatinase activity and pellicle formation in extended-spectrum beta-lactamase (ESBL) and non-ESBL producing Acinetobacter baumannii isolates from diabetic foot ulcer infection (DFI).Methods: A total of 42 isolates of A. baumannii recovered from patients of DFI from September 2016 to February 2018. Isolates were identified by the standard microbiological method and confirmed by the BD Phoenix 100 system. The antimicrobial susceptibility test was performed by the Kirby–Bauer disk diffusion method and ESBL was detected by double disk diffusion synergy test method. Gelatinase production was determined by the Luria Bertani agar supplemented with 30 g/L gelatin, and pellicle formation was determined by the Mueller-Hinton broth which is incubated at two different temperatures.Results: A total of 42 A. baumannii isolates were multidrug resistant. Among 21 isolates, each was ESBL and non-ESBL producers. Pellicle formation at 25°C in ESBL and non-ESBL producer isolates was 47.61% (10/21) and 28.57% (06/21). Pellicle formation at 37°C in ESBL and non-ESBL producer isolates was 57.14% (12/21) and 42.85% (09/21), respectively. Gelatinase production was present in 38.09% ESBL and 28.57% in non-ESBL producers. ESBL strains were more virulent compared to non-ESBL producers among patients of DFIs.Conclusion: This study showed that pellicle formation at 37°C was highly virulent due to ESBL producers. Gelatinase production was elevated in ESBL compared to non-ESBL producer isolates. This attribute of the isolates could render ESBL positive more pathogenic. Colistin and polymyxin B are the only choices of treatment for multidrug-resistant Acinetobacter baumannii infections.

Mcr colistin resistance gene: a systematic review of current diagnostics and detection methods

Resistance to colistin, mediated by chromosomal mutations and more recently, by plasmid‐borne mcr genes, is increasingly being reported in bacterial isolates taken from humans, animals, farms, foods, and the environment. To easily identify and contain this quickly spreading menace, efficient diagnostics that are cheaper, faster, simpler, sensitive, and specific have become indispensable and urgently necessary. A thorough and systematic review of the literature available at Pubmed, ScienceDirect and Web of Science was thus undertaken to identify articles describing novel and efficient colistin resistance‐ and mcr gene‐detecting methods. From the final 23 studies included in this review, both phenotypic and molecular tests were found. The phenotypic tests consisted of novel culture media viz., SuperPolymyxin™, CHROMagar COL‐APSE and LBJMR media, commercial automated MIC‐determining instruments such as MICRONAUT‐S, Vitek 2, BD Phoenix, Sensititre and MicroScan, and novel assays such as Colistin MAC test, Colispot, rapid polymxin NP test (RPNP), alteration of Zeta potential, modified RPNP test, MICRONAUT‐MIC Strip, MIC Test Strip, UMIC System, and Sensitest™ Colistin. Molecular diagnostics consisted of the CT103XL microarray, eazyplex® SuperBug kit, and Taqman®/SYBR Green® real‐time PCR assays, with 100% sensitivity and specificity plus a shorter turnaround time (<3 hr). Based on the sensitivity, specificity, cost, required skill and turnaround time, the RPNP test and/or novel culture media is recommended for under‐resourced laboratories while the Multiplex PCR or Taqman®/SYBR Green® real‐time PCR assay alongside the RPNP or novel culture media is suggested for well‐resourced ones.

Microbiology and clinical characteristics of viridans group streptococci in patients with cancer

This study assessed the microbiology, clinical syndromes, and outcomes of oncologic patients with viridans group streptococci isolated from blood cultures between January 1st, 2013 and December 31st, 2016 in a referral hospital in Mexico using the Bruker MALDI Biotyper. Antimicrobial sensitivity was determined using BD Phoenix 100 according to CLSI M100 standards. Clinical information was obtained from medical records and descriptive analysis was performed.Forty-three patients were included, 22 females and 21 males, aged 42 ± 17 years. Twenty (46.5%) patients had hematological cancer and 23 (53.5%) a solid malignancy. The viridans group streptococci isolated were Streptococcus mitis, 20 (46.5%); Streptococcus anginosus, 14 (32.6%); Streptococcus sanguinis, 7 (16.3%); and Streptococcus salivarius, 2 (4.7%). The main risk factors were pyrimidine antagonist chemotherapy in 22 (51.2%) and neutropenia in 19 (44.2%) cases, respectively. Central line associated bloodstream infection was diagnosed in 18 (41.9%) cases. Septic shock occurred in 20.9% of patients, with an overall mortality of 18.6%. Only four S. mitis revealed penicillin-resistance.Our results are similar to those of other series, identifying these bacteria as emerging pathogens with significant morbidity and mortality in oncologic patients. The MALDI-TOF system increased the rate of viridans group streptococci isolation in this population.

Visible DNA Microarray System as an Adjunctive Molecular Test in Identification of Pathogenic Fungi Directly from a Blood Culture Bottle

A DNA microarray platform, based on the nucleotide sequences of the internal transcribed spacer regions (ITS1 and ITS2) of the rRNA gene, was developed to identify 32 fungal pathogens at the species level. The probe sequences were spotted onto polycarbonate slides with a mini-microarray printer, and after the hybridization, the results were visible with the naked eye. The performance of the microarray platform was evaluated against the commercial automated systems (Vitek 2 and BD Phoenix systems) and DNA sequencing (gold standard). A total of 461 blood culture bottles were tested: 127 positive for fungi, 302 positive for bacteria, and 32 that were negative. Once the microorganisms were identified by automated systems, fungal DNA was extracted directly from the blood culture bottles. The DNA products were tested using the microarray platform, and DNA sequencing was performed. The results of the microarray and DNA sequencing were concordant in 96.7% of cases, and the results from the automated systems and DNA sequencing were concordant in 98.4%. Of all the nucleotide sequences contained in the microarray platform, the microarray failed to identify four fungal isolates (one Candida parapsilosis, two Candida tropicalis, and one Cryptococcus neoformans). Of note, the microarray detected Candida krusei DNA in two blood cultures from the same patient, whereas the automated system was only positive for Enterococcus faecium Our microarray system provided reliable and fast fungal identification compared to that from DNA sequencing and the automated systems. The simplicity of reading the results by the naked eye made this DNA platform a suitable method for fungal molecular diagnosis.

Comparation of Phenotypic and Genotypic Profile of Carbapenemase Producing Escherichia coli

Carbapenemase-producing Escherichia coli (E. coli) has caused trouble in therapeutic antibiotic selection. Carbapenemase screening procedure in laboratories is usually based on inacurate semi-automatic system. Confirmation and classification of carbapenemases according to Ambler can be done with combination of phenotypic methods, i.e., Modified Hodge Test (MHT), Sodium Mercaptoacetic Acid (SMA), and 3-Aminophenylboronic Acid (PBA). This study aimed to compare profiles of carbapenemase-producing E. coli which were confirmed and classified phenotypically with the genotypic profiles. E. coli isolates from urine specimens which were potential as carbapenemase-producers according to semi-automatic system BD Phoenix were phenotypically tested with MHT, SMA, and PBA. Isolates were grouped as carbapenemase-producers and non carbapenemase-producers. Phenotypic carbapenemase-producer isolates were classified based on Ambler criteria. All isolates were then tested with Polymerase Chain Reaction (PCR) for the presence of OXA-48, IMP1, IMP2, GES, VIM, NDM, KPC genes. Out of 30 isolates, 6 isolates (20.0%) were MHT positive, and 25 isolates (83.3%) were SMA positive, which indicated that most isolates produced were carbapenemase Ambler B. PCR confirmed 12 isolates (40.0%) had VIM gene which were classified as carbapenemase Ambler B. Phenotypic confirmatory test had 100% sensitivity and 22.2% specificity. Classification with phenotypic confirmatory test had 91.7% match with PCR. Phenotypic confirmatory test detected more carbapenemase than PCR. This low specificity may be caused by inappropriate use of diagnostic gold standard. PCR should not be used for routine carbapenemase confirmation because of vast diversity of carbapenemases. Phenotypic confirmatory test can classify carbapenemase according to Ambler classification.

CHARACTERIZATION OF VARIOUS VIRULENCE PROPERTIES OF DRUG-RESISTANT KLEBSIELLA ISOLATES: AN IN VITRO STUDY AMONG “SUPERBUGS”

Objective: The objective of this study was to evaluate the various virulence factors among all extended-spectrum beta-lactamase (ESBL)-producing Klebsiella isolates.Methods: Klebsiella species (n=201) isolated from various non-repeated clinical samples from October 2016 to May 2017 were collected and identified by biochemical method and confirmed by BD Phoenix. Antibiotic susceptibility test was performed by Kirby-Bauer disc diffusion method, and ESBL detection was done by combined disc diffusion method. Capsule detection was done by nigrosin dye staining, hypermucoviscosity was done by string test, biofilm was performed by microtiter plate assay, and hemagglutination was performed with human erythrocytes treated with tannic acid.Results: Among 201 ESBL-producing Klebsiella isolates, 63.2 % were multidrug resistance and 24.4% were extensively drug-resistant. Among these isolates, Klebsiella infection is more in older patients. All isolates were (100%) positive for capsule and siderophores production. Of all 201 isolates, 14 (7%) were strong, 61 (30.3%) were moderate, and 104 (51.8%) were weak biofilm producers. Among these isolates, 36 (17.9%) were type 1 fimbriae, 140 (69.7%) were type 3 fimbriae, and only 17 (8.5%) were positive for hypermucoviscosity.Conclusion: Several virulence factors were studied, in which biofilm with fimbriae is the main cause for the resistance of antibiotics. Bacterial resistance profile found to be high with ESBL production and multiple drug resistance in the majority of the Klebsiella isolates, which is a result of improper antibiogram schedule and their virulence property.

GENOTYPIC AND PHENOTYPIC CHARACTERIZATION OF Alcaligenes javaensis JG3 POTENTIAL AS AN EFFECTIVE BIODEGRADER

Utilization of glycerol by lipase producing bacteria offers great benefits for fat and oil waste degradation and waterwaste treatment. Nevertheless, there have been lack of reports about the availability of non-pathogenic, lipase producing bacteria, which could naturally degrade glycerol produced from the lipolysis process by lipase. This study reported a newly identified species of rhizobacteria, Alcaligenes javaensis JG3, which is not only able to produce high level of lipase, but also able to degrade glycerol molecules. Identification of strain JG3 was carried out using SEM (Scanning Electron Microscope), BD Phoenix 100 Automated Microbiology System and 16S rRNA gene analysis to determine its taxonomy status. The ability of the strain to metabolize glycerol was investigated both genotypically and phenotypically using degenerate PCR and a glycerol minimal medium. Identification test results showed that strain JG3 belongs to genus Alcaligenes, with the closest relationship with A. faecalis and A. aquatilis (96% nucleotide similarity maximum). Degenerate PCR resulted in a 248-bp sequence showing 93% similarity with glpK of Candidatus Sodalis pierantonius SOPE, a key gene involved in glycerol metabolism. In vitro glycerol utilization test result showed that Alcaligenes sp. JG3 was able to grow on glycerol aerobically, but not anaerobically. It is concluded that Alcaligenes sp. JG3 possesses genes coding for glycerol metabolism and this trait is phenotypically expressed, thus making the strain potential to be used as an effective fat and oil biodegrader.

Comparison of tigecycline susceptibility testing methods for multidrug-resistant Acinetobacter baumannii

Automated systems Vitek 2, BD Phoenix, MicroScan WalkAway and gradient diffusion assays Etest and MIC Test Strip were compared against broth microdilution for susceptibility testing of tigecycline against Acinetobacter baumannii. BD Phoenix and MIC Test Strip seemed to provide the most accurate determination of tigecycline susceptibility.

Multidrug resistance E-ESKAPE strains isolated from blood cultures in patients with cancer

To describe the trend of multidrug resistant (MDR) strains isolated from blood in patients with cancer from 2005 to 2015. 33 127 blood cultures were processed by retrospective analysis. Identification and antimicrobial sensitivity were performed through automated methods: WaLK away (Siemens Labora- tory Diagnostics) and BD Phoenix (Becton, Dickinson and Company). Resistant strains were determined according to the minimum inhibitory concentration, following the parameters of the Clinical and Laboratory Standards Institute (CLSI). Of 6 397 isolates, 5 604 (16.9%) were positive; 3 732 (58.4%) Gram- bacilli; 2 355 (36.9%) Gram+ cocci; 179 (2.7%) yeasts, and 126 (1.9%) Gram+ bacilli. Escherichia coli (n=1 591, 24.5%) was the most frequent bacteria, with 652 (41%) strains being extended-spectrum beta-lactamases producers (ESBL); of Enterococcus faecium (n=143, 2.1%), 45 (31.5%) were vancomycin resistant; of Staphylococcus aureus (n=571, 8.7%), 121 (21.2%) methicillin resistant (MRSA); of Klebsiella pneumoniae (n=367, 5.6%), 41 (11.2%) ESBL; of Acinetobacter baumanii (n=96, 1.4%), 23 (24%) MDR, and of Pseudomonas aeruginosa (n=384, 5.6%), 43 (11.2%) MDR. MDR strains were significantly more frequent in patients with hematological malignancies, compared to those with solid tumors: MRSA (OR=4.48, 95%CI 2.9-6.8), ESBL E. coli(OR=1.3, 95%CI 1.10-1.65) and MDR Acinetobacter baumanii (OR=3.2, 95%CI 1.2-8.3). We observed significantly higher isolations of E-ESPAKE MDR strains in patients with hematological malignancies.

The era of device colonizers:Chryseobacterium indologenesinfections from a tertiary care center in North India

Background:Chryseobacterium indologenes is a hospital environment contaminant and can cause healthcare-associated infections.Methods:Patients with C. indologenes infections in a tertiary care center in North India for 6 months were evaluated for susceptibility patterns, comorbidities, mechanical devices, risk factors, and treatment outcomes. The organism was provisionally identified phenotypically, and identification was confirmed by the BD Phoenix automated microbiology system. Minimum inhibitory concentration values of antibiotic susceptibility were determined.Results:A total of 12 isolates of C. indologenes were recovered from 11 patients. Five patients had C. indologenes bloodstream infection (BSI), one had ventilator-associated pneumonia (VAP), and one had both BSI and VAP. In four others, the organism was isolated from the catheterized urinary tract. All VAP and BSI patients were admitted to the Intensive Care Units and mechanically ventilated; all had central lines and history of colistin therapy during the past 15 days. The common underlying risk factors were diabetes, hypertension, and coronary artery disease.Conclusions:C. indologenes infections are increasing because of higher use of carbapenems and colistin, to which it is intrinsically resistant.

Transformation From a Conventional Clinical Microbiology Laboratory to Full Automation.

To validate the performance, reproducibility, and reliability of BD automated instruments in order to establish a fully automated clinical microbiology laboratory. We used control strains and clinical samples to assess the accuracy, reproducibility, and reliability of the BD Kiestra WCA, the BD Phoenix, and BD Bruker MALDI-Biotyper instruments and compared them to previously established conventional methods. The following processes were evaluated: sample inoculation and spreading, colony counts, sorting of cultures, antibiotic susceptibility test, and microbial identification. The BD Kiestra recovered single colonies in less time than conventional methods (e.g. E. coli, 7h vs 10h, respectively) and agreement between both methodologies was excellent for colony counts (κ=0.824) and sorting cultures (κ=0.821). Antibiotic susceptibility tests performed with BD Phoenix and disk diffusion demonstrated 96.3% agreement with both methods. Finally, we compared microbial identification in BD Phoenix and Bruker MALDI-Biotyper and observed perfect agreement (κ=1) and identification at a species level for control strains. Together these instruments allow us to process clinical urine samples in 36h (effective time). The BD automated technologies have improved performance compared with conventional methods, and are suitable for its implementation in very busy microbiology laboratories.

In vitro effect of vancomycin and daptomycin on biofilm formation of coagulase-negative staphylococci strains

Coagulase-negative staphylococci (CNS) are one of the primer agents of blood stream infections (BSI) and catheter-related bloodstream infections (CR-BSI) which are associated mostly with the usage of central venous catheters and, important causes of morbidity and mortality despite the usage of antibacterial and supportive treatment. It is important to determine the properties of these causative microorganisms in order to make appropriate treatment of such infections. The aims of our study were to evaluate the biofilm formation of coagulase negative staphylococci (CNS) which were causative agents of bloodstream (BSI) and catheter related bloodstream infections (CR-BSI), to determine the minimum inhibitory concentration (MIC) of planktonic forms and minimal biofilm eradication concentration (MBEC) of sessile forms for vancomycin and daptomycin and to evaluate the efficacy of these antibiotics in infections with biofilm-forming isolates in vitro. A total of 65 CoNS (n= 26 catheter colonizers, n= 28 CR-BSI, n= 11 BSI agents) were identified by conventional methods and also with BD Phoenix (Becton Dickinson, USA) and Bruker Microflex MS (Bruker Daltonics, Germany) systems. Methicillin resistance was determined by the presence of mecA gene with PCR. MIC values of vancomycin and daptomycin were investigated by broth microdilution, for daptomycin medium containing 25 and 50 μg/ml Ca++ were used. Assessment of biofilm formation and detection of MBEC were determined by microplate method. The clonal relationship was investigated by the PFGE method. A total of 65 isolates; 26 catheter colonizers, 28 CR-BSI agents and 11 BSI agents were evaluated and identified as Staphylococcus epidermidis (n= 33), Staphylococcus haemolyticus (n= 16), Staphylococcus hominis (n= 15), and Staphylococcus capitis (n= 1). 81.5% of the isolates were found to be methicillin resistant and all of them were found to be sensitive to vancomycin (MIC= 0.125-4 μg/ml) and daptomycin (MIC= 0.062-0.25 μg/ml in 25 μg/ml Ca++ and MIC= 0.031-0.50 μg/ml in 50 μg/ml Ca++ containing medium). MIC values were lower in medium containing 50 μg/ml Ca++ for daptomycin. As it is known that the efficacy of daptomycin depends on the physiological levels of Ca++, which causes conformational changes in the structure of these antibacterials. Our findings also suggested that high levels of Ca++ are needed to ensure the efficacy of daptomycin. All of the isolates produced biofilm at different strengths of positivity (n= 12/18.5% weak, n= 35/%53.8 moderate, n= 18/%27.7 strong). MBEC and MBEC/MIC values for vancomycin were found to be higher than daptomycin (p< 0.001). Strong biofilm producers had higher MBEC and MBEC/MIC, MBEC50/MIC50 ve MBEC90/MIC90 values (p< 0.05). Especially in infections with biofilm forming isolates, the detection of only MIC values are not always sufficient in the treatment of biofilm-related infections as they reflect the sensitivity of planktonic bacteria. The inconsistency between the MIC and MBEC values and the high rates of MBEC/MIC found in our study supported this prediction.The lower detection of MBEC and MBEC/MIC values of daptomycin compared to the same values of vancomycin suggested that daptomycin might be effective at lower doses than vancomycin in the treatment of biofilm infections.

Discriminatory Power of Three Typing Techniques in Determining Relatedness of Nosocomial Klebsiella pneumoniae Isolates from a Tertiary Hospital in India

Purpose: The purpose of this study was to evaluate the discriminatory power of two DNA-based technique and a protein-based technique for the typing of nosocomial isolates of Klebsiella pneumoniae. A second objective was to determine the antimicrobial susceptibility pattern and characterise the presence of genes encoding extended-spectrum beta-lactamases (ESBLs) and carbapenemases. Materials and Methods: Forty-six K. pneumoniae isolates from patients with bloodstream infections at a tertiary care hospital in India between December 2014 and December 2015 were studied. All isolates were typed using enterobacterial repetitive intergenic consensus sequence-polymerase chain reaction (ERIC-PCR), randomly amplified polymorphic DNA (RAPD) analysis and matrix-assisted laser desorption ionisation time-of-flight (MALDI-TOF) mass spectrometry. Antimicrobial susceptibility profiles and ESBLs were detected using the BD Phoenix system. The types of ESBL and carbapenemase genes present were detected using PCR. Results: Isolates were subtyped into 31, 30 and 33 distinct genotypes by ERIC-PCR, RAPD and MALDI-TOF, respectively. Several isolates displaying identical DNA fingerprints were binned into different branches based on their proteomic fingerprint. Antimicrobial susceptibility tests revealed that 33/46 strains were multidrug resistant (MDR); a majority of the strains (83%) were sensitive to colistin. PCR-based analysis demonstrated 19 strains to harbour two or more ESBL and carbapenemase genes. Conclusion: ERIC-PCR was the most reproducible method for typing K. pneumoniae isolates and could not be substituted by MALDI-TOF for clonality analysis. A high degree of genetic diversity and the presence of MDR genes highlight the challenges in treating K. pneumoniae-associated infections.

Comparison of methods for detection of plasmid-mediated and chromosomally encoded colistin resistance in Enterobacteriaceae

ObjectivesBecause of the emergence of plasmid-mediated (mcr-1 and mcr-2 genes) and chromosomally encoded colistin resistance, reliable methods for detecting colistin resistance/susceptibility in routine laboratories are required. We evaluated the respective performances of the BD Phoenix automated system, the newly developed Rapid Polymyxin NP test and the broth microdilution (BMD) reference method to detect colistin resistance in Enterobacteriaceae, and particularly those producing mcr-1 and mcr-2. MethodsColistin susceptibility of 123 enterobacterial clinical isolates (40 colistin-susceptible and 83 colistin-resistant isolates) was tested with the BD Phoenix automated system, the Rapid Polymyxin NP test and the BMD method. Molecular mechanisms responsible for plasmid-mediated and chromosomally encoded colistin resistance mechanisms were investigated by PCR and sequencing. ResultsConsidering BMD as a reference method, the BD Phoenix system failed to detect ten colistin-resistant isolates (one Escherichia coli, one Klebsiella pneumoniae, seven Enterobacter species and one Salmonella enterica). The Rapid Polymyxin NP test failed to detect the same single E. coli isolate. Those two latter methods detected the 16 E. coli, K. pneumoniae and S. enterica isolates producing the plasmid-encoded mcr-1 and mcr-2. ConclusionsThe BD Phoenix system and the Rapid Polymyxin NP test are reliable techniques for detecting plasmid-mediated mcr-1 and mcr-2-related colistin resistance. However, a high rate of false susceptibility was observed with the BD Phoenix system, indicating that susceptibility results obtained with that system should be confirmed by BMD method. By contrast, the Rapid Polymyxin NP test showed a good agreement with the BMD method, and results were obtained rapidly (within 2 hours). The BMD method should be performed if minimum inhibitory concentration values are needed.